scholarly journals Investigation of the forms of pig duodenal Ca2+-binding protein produced by limited tryptic hydrolysis

1986 ◽  
Vol 237 (3) ◽  
pp. 781-787 ◽  
Author(s):  
D T W Bryant ◽  
S Critch
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Binding Protein ◽  
Native Protein ◽  
Binding Studies ◽  
High Affinity ◽  
C Terminus ◽  
Kd Values ◽  
T1 And T2 ◽  
Tryptic Hydrolysis

Vitamin D-dependent Ca2+-binding protein from pig duodenum was hydrolysed with trypsin in the presence of Ca2+ and two products were obtained: T1, which differed from the native protein by loss of Ac-Ser-Ala-Gln-Lys from the N-terminus and Ile-Ser-Gln-OH from the C-terminus, and T2, which differed from T1 by loss of a C-terminal lysine. The hydrolysis inactivated one of the two high-affinity Ca2+-binding sites on the native protein, and the remaining site was stable in T1 but labile in T2 when the proteins were Ca2+-free. Binding studies showed that T1 had Kd values of 2.8 +/- 0.1 nM, 57 +/- 13 microM and 0.8 +/- 0.3 microM for Ca2+, Mg2+ and Mn2+ respectively, and T2 had Kd 2.2 +/- 0.3 nM for Ca2+. The affinity for Mn2+, together with the other Kd values, identified the site on T1 as the site on the native protein previously found to have Kd 0.6 microM for Mn2+, rather than one with Kd 50 microM for Mn2+. In contrast with both the native protein and another form of the protein with a single Ca2+-binding site, the intrinsic fluorescence of T1 and T2 was little affected by the addition of Ca2+. It was concluded that the active binding site in T1 and T2, and also the site in the native protein with the higher affinity for Mn2+, was probably in the C-terminal half of the molecule.

scholarly journals Molecular target sizes of inositol 1,4,5-trisphosphate receptors in liver and cerebellum

1990 ◽  
Vol 265 (2) ◽  
pp. 393-398 ◽  
Author(s):  
D L Nunn ◽  
B V L Potter ◽  
C W Taylor
Keyword(s):  
Binding Sites ◽  
Radioligand Binding ◽  
Inositol Phosphate ◽  
Rank Order ◽  
Binding Protein ◽  
Molecular Target ◽  
Binding Studies ◽  
Single Class ◽  
Surface Receptors ◽  
High Affinity

Ins(1,4,5)P3 is the intracellular messenger that mediates the effects of many cell-surface receptors on intracellular Ca2+ stores. Although radioligand-binding studies have identified high-affinity Ins(1,4,5)P3-binding sites in many tissues, these have not yet been convincingly shown to be the receptors that mediate Ca2+ mobilization, nor is it clear whether there are differences in these binding sites between tissues. Here we report that Ins(1,4,5)P3 binds to a single class of high-affinity sites in both permeabilized hepatocytes (KD = 7.8 +/- 1.1 nM) and cerebellar membranes (KD = 6.5 +/- 2.4 nM), and provide evidence that these are unlikely to reflect binding to either of the enzymes known to metabolize Ins(1,4,5)P3. Furthermore, the rank order of potency of synthetic inositol phosphate analogues in displacing specifically bound Ins(1,4,5)P3 is the same as their rank order of potency in stimulating mobilization of intracellular Ca2+ stores, suggesting that the Ins(1,4,5)P3-binding site may be the physiological receptor. Radiation inactivation of the Ins(1,4,5)P3-binding sites of liver and cerebellum reveals that they have similar molecular target sizes: 257 +/- 36 kDa in liver and 258 +/- 20 kDa in cerebellum. We conclude that an Ins(1,4,5)P3-binding protein with a molecular target size of about 260 kDa is probably the receptor that mediates Ca2+ mobilization in hepatocytes, and our limited data provide no evidence to distinguish this from the cerebellar Ins(1,4,5)P3-binding protein.

scholarly journals Liver inositol, 1,4,5-trisphosphate-binding sites are the Ca2(+)-mobilizing receptors

1990 ◽  
Vol 270 (1) ◽  
pp. 227-232 ◽  
Author(s):  
D L Nunn ◽  
C W Taylor
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Maximal Effect ◽  
Equilibrium Conditions ◽  
Peripheral Tissues ◽  
High Affinity ◽  
Kd Values ◽  
Release Data ◽  
The Relationship ◽  
Similar Affinity

Ins(1,4,5)P3 is the intracellular messenger that in many cells mediates the effects of Ca2(+)-mobilizing receptors on intracellular Ca2+ stores. An Ins(1,4,5)P3 receptor from cerebellum has been purified and functionally reconstituted, but the relationship between this protein and the high-affinity Ins(1,4,5)P3-binding sites of peripheral tissues is unclear. We compared the Ins(1,4,5)P3-binding sites of liver and cerebellum by measuring inhibition of specific Ins(1,4,[32P]5)P3 binding by various ligands under equilibrium conditions, and find that each ligand binds with similar affinity in the two tissues. Earlier studies in which Ins(1,4,5)P3 binding and Ca2+ mobilization were measured under different conditions demonstrated large differences between KD values for binding and EC50 values (concn. giving half-maximal effect) for Ca2+ release. We show here that, when measured under identical conditions, KD and EC50 values for four agonists are similar. Schild analysis of inhibition of Ins(1,4,5)P3 binding by ATP demonstrates a competitive interaction between the two at the liver Ins(1,4,5)P3-binding site, and this partly accounts for earlier discrepancies in binding and Ca2(+)-release data. We conclude that the high-affinity Ins(1,4,5)P3-binding site of hepatocytes is likely to be the receptor that mediates Ca2+ mobilization, and that this receptor is at present indistinguishable from that in cerebellum.

The somatotrophic axis in young steers: influence of nutritional status and oestradiol-17β on hepatic high-and low-affinity somatotrophic binding sites

1988 ◽  
Vol 116 (2) ◽  
pp. 169-177 ◽  
Author(s):  
B. H. Breier ◽  
P. D. Gluckman ◽  
J. J. Bass
Keyword(s):  
Body Weight ◽  
Nutritional Status ◽  
Binding Site ◽  
Binding Sites ◽  
Dry Matter ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Binding Studies ◽  
High Affinity ◽  
High Affinity Site

ABSTRACT The binding of bovine GH (bGH) to hepatic membranes obtained from steers on either high (3% dry matter of body weight per day) or low (1% dry matter of body weight per day) planes of nutrition with or without an oestradiol-17β implant was studied (n = 5 per group). Binding studies were performed on both crude membrane homogenates and on 100 000 g microsomal membrane fractions; identical results were obtained using both preparations. In all four groups of animals, linear Scatchard plots were obtained, but following pretreatment of the membranes with MgCl2 to remove endogenously bound hormone, curvilinear plots were obtained in the groups on the high plane of nutrition. Analysis of these curves suggested the presence of a high- and low-affinity binding site, the high-affinity site being fully occupied in the absence of MgCl2 pretreatment. The specific binding of bGH in MgCl2-pretreated crude membranes was greater (P < 0·01) in well-fed steers (14·8 ± 1·6%) than in poorly fed steers (9·8 ± 0·9%). Scatchard analysis showed this to be due to the presence of a high-affinity site (dissociation constant (Kd) = 11·6 ± 3·3 pmol/l) in the well-fed animals only. In addition, there was an increase (P < 0·01) in the affinity, but not in the capacity, of the low-affinity site (Kd = 106·4 ± 22·8 pmol/l in well-fed steers and 197·0 ± 23·8 pmol/l in poorly fed steers). Oestradiol treatment was associated with an increase (P < 0·01) in specific binding at both planes of nutrition, but binding was higher (P < 0·01) in well-fed (24·8 ± 2·9%) than in poorly fed (15·6 ± 3·7%) steers. Scatchard analysis after MgCl2 pretreatment again showed a curvilinear plot at the high and a linear plot at the low nutritional plane. The effect of oestradiol was to increase (P < 0·001) the capacity of the high-affinity site from 1·87 ± 0·61 pmol/100 mg in the control well-fed group to 6·56 ± 1 ·2 pmol/100 mg. The capacity of the low-affinity site was increased (P < 0·01) from 20·1 ± 2·6 to 30·1 ± 3·2 pmol/100 mg in the well-fed group, with a similar change in the poorly fed group. Oestradiol had no effect on the apparent affinity of either binding site. These studies demonstrate a heterogeneity of somatotrophic binding sites of hepatic membranes in steers. The presence of a high-affinity site is determined by nutritional status, whereas oestradiol primarily affects receptor capacity. Thus nutrition and oestradiol have independent and qualitatively different effects on somatotrophic binding. As the rate of weight gain correlated (P < 0·01) with the capacity of the high-affinity site, it is suggested that somatotrophic receptor modulation is a primary factor in the regulation of somatic growth in the ruminant. J. Endocr. (1988) 116, 169–177

scholarly journals Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing

1992 ◽  
Vol 12 (3) ◽  
pp. 1209-1217
Author(s):  
C F Hardy ◽  
D Balderes ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Binding Protein ◽  
Binding Domain ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Carboxy Terminal

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals [3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

1992 ◽  
Vol 40 (6) ◽  
pp. 771-779 ◽  
Author(s):  
A A Maki ◽  
D G Baskin ◽  
W L Stahl
Keyword(s):  
Nervous System ◽  
Rat Brain ◽  
Cardiac Glycoside ◽  
Binding Sites ◽  
Quantitative Autoradiography ◽  
Affinity Binding ◽  
Ouabain Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Kd Values

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.

scholarly journals Progesterone Receptor Membrane Component-1 (PGRMC1) Is the Mediator of Progesterone’s Antiapoptotic Action in Spontaneously Immortalized Granulosa Cells As Revealed by PGRMC1 Small Interfering Ribonucleic Acid Treatment and Functional Analysis of PGRMC1 Mutations

Endocrinology ◽  
2007 ◽  
Vol 149 (2) ◽  
pp. 534-543 ◽  
Author(s):  
John J. Peluso ◽  
Jonathan Romak ◽  
Xiufang Liu
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Transmembrane Domain ◽  
Binding Protein ◽  
Plasminogen Activator Inhibitor ◽  
Membrane Receptor ◽  
Membrane Component ◽  
Plasminogen Activator Inhibitor 1 ◽  
Receptor Membrane ◽  
C Terminus

Progesterone (P4) receptor membrane component-1 (PGRMC1) and its binding partner, plasminogen activator inhibitor 1 RNA binding protein (PAIRBP1) are thought to form a complex that functions as membrane receptor for P4. The present investigations confirm PGRMC1’s role in this membrane receptor complex by demonstrating that depleting PGMRC1 with PGRMC1 small interfering RNA results in a 60% decline in [3H]P4 binding and the loss of P4’s antiapoptotic action. Studies conducted on partially purified GFP-PGRMC1 fusion protein indicate that [3H]P4 specifically binds to PGRMC1 at a single site with an apparent Kd of about 35 nm. In addition, experiments using various deletion mutations reveal that the entire PGRMC1 molecule is required for maximal [3H]P4 binding and P4 responsiveness. Analysis of the binding data also suggests that the P4 binding site is within a segment of PGRMC1 that is composed of the transmembrane domain and the initial segment of the C terminus. Interestingly, PAIRBP1 appears to bind to the C terminus between amino acids 70–130, which is distal to the putative P4 binding site. Taken together, these data provide compelling evidence that PGRMC1 is the P4 binding protein that mediates P4’s antiapoptotic action. Moreover, the deletion mutation studies indicate that each domain of PGRMC1 plays an essential role in modulating PGRMC1’s capacity to both bind and respond to P4. Additional studies are required to more precisely delineate the role of each PGRMC1 domain in transducing P4’s antiapoptotic action.

Generation of an agonistic binding site for blockers of the M3 muscarinic acetylcholine receptor

2008 ◽  
Vol 412 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Doreen Thor ◽  
Angela Schulz ◽  
Thomas Hermsdorf ◽  
Torsten Schöneberg
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
G Protein ◽  
Binding Sites ◽  
Expression System ◽  
Muscarinic Acetylcholine Receptor ◽  
Intracellular Loop ◽  
Inverse Agonists ◽  
Yeast Expression System ◽  
High Affinity

GPCRs (G-protein-coupled receptors) exist in a spontaneous equilibrium between active and inactive conformations that are stabilized by agonists and inverse agonists respectively. Because ligand binding of agonists and inverse agonists often occurs in a competitive manner, one can assume an overlap between both binding sites. Only a few studies report mutations in GPCRs that convert receptor blockers into agonists by unknown mechanisms. Taking advantage of a genetically modified yeast strain, we screened libraries of mutant M3Rs {M3 mAChRs [muscarinic ACh (acetylcholine) receptors)]} and identified 13 mutants which could be activated by atropine (EC50 0.3–10 μM), an inverse agonist on wild-type M3R. Many of the mutations sensitizing M3R to atropine activation were located at the junction of intracellular loop 3 and helix 6, a region known to be involved in G-protein coupling. In addition to atropine, the pharmacological switch was found for other M3R blockers such as scopolamine, pirenzepine and oxybutynine. However, atropine functions as an agonist on the mutant M3R only when expressed in yeast, but not in mammalian COS-7 cells, although high-affinity ligand binding was comparable in both expression systems. Interestingly, we found that atropine still blocks carbachol-induced activation of the M3R mutants in the yeast expression system by binding at the high-affinity-binding site (Ki ∼10 nM). Our results indicate that blocker-to-agonist converting mutations enable atropine to function as both agonist and antagonist by interaction with two functionally distinct binding sites.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals The C Terminus of the Major Yeast Telomere Binding Protein Rap1p Enhances Telomere Formation

1998 ◽  
Vol 18 (3) ◽  
pp. 1284-1295 ◽  
Author(s):  
Alo Ray ◽  
Kurt W. Runge
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Binding Protein ◽  
Telomerase Activity ◽  
Telomerase Rna ◽  
Constant Length ◽  
C Terminus ◽  
Telomere Binding Protein ◽  
Yeast Telomere

ABSTRACT The telomeres of most organisms consist of short repeated sequences that can be elongated by telomerase, a reverse transcriptase complex that contains its own RNA template for the synthesis of telomere repeats. In Saccharomyces cerevisiae, the RAP1gene encodes the major telomere binding protein Rap1p. Here we use a quantitative telomere formation assay to demonstrate that Rap1p C termini can enhance telomere formation more than 30-fold when they are located at internal sites. This stimulation is distinct from protection from degradation. Enhancement of formation required the gene for telomerase RNA but not Sir1p, Sir2p, Sir3p, Sir4p, Tel1p, or the Rif1p binding site in the Rap1p C terminus. Our data suggest that Rap1p C termini enhance telomere formation by attracting or increasing the activity of telomerase near telomeres. Earlier work suggests that Rap1p molecules at the chromosome terminus inhibit the elongation of long telomeres by blocking the access of telomerase. Our results suggest a model where a balance between internal Rap1p increasing telomerase activity and Rap1p at the termini of long telomeres controlling telomerase access maintains telomeres at a constant length.

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