scholarly journals An H.P.L.C. Study of Cooperative Guest Binding by a Covalently Linked b-Cyclodextrin Dimer

1997 ◽  
Vol 50 (8) ◽  
pp. 857 ◽  
Author(s):  
Anna K. Croft ◽  
Christopher J. Easton ◽  
Stephen F. Lincoln ◽  
Bruce L. May ◽  
John Papageorgiou
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
High Performance ◽  
Association Constant ◽  
Association Constants ◽  
Cooperative Binding ◽  
Cyclodextrin Dimer ◽  
Guest Binding ◽  
Covalently Linked ◽  
Single Binding Site

The complexation of the anions of Methyl Orange (MO¯) (1) and Indomethacin (Ind-) (2) by the β-cyclodextrin dimer (3) has been studied by means of high-performance liquid chromatography. The cyclodextrin annuli are shown to jointly act as a single binding site for complexation of MO¯ (1). Thus the association constants of (3·3±0·3) × 103 and (220±20) × 103 dm3 mol−1 for the 1 : 1 complexes with β-cyclodextrin and the dimer (3), respectively, indicate very strong cooperative binding by the annuli of the linked species. By contrast, the complexation of Ind¯ (2) by the cyclodextrin dimer (3) is characterized by two independent binding sites. This lack of cooperative binding is reflected in the association constant for the 1 : 1 complex in each binding site, which at (1·3±0·1) × 103 dm3 mol−1 is little greater than that of (0·7±0·05) × 103dm3 mol−1 for the complex with β-cyclodextrin.

Binding of copper(II) ion to cytochrome c

1981 ◽  
Vol 34 (1) ◽  
pp. 91 ◽  
Author(s):  
MA Augustin ◽  
JK Yandell
Keyword(s):  
Ionic Strength ◽  
Cytochrome C ◽  
Binding Site ◽  
Spectral Data ◽  
Binding Sites ◽  
Association Constant ◽  
Association Constants ◽  
Visible Absorption ◽  
Ph Range ◽  
Horse Cytochrome

Binding of aquacopper(11) ion to horse and tuna ferricytochrome c has been investigated over the pH range 2-9. Between pH 4 and 7 the principal binding site for horse cytochrome has an intrinsic association constant of 1.8 × 104 1. mol-1 and pKa of 6.5�0.6 at 25�C and ionic strength of 0.1. Binding to histidine 33 is consistent with this information, as well as with e.s.r., n.m.r. and visible absorption spectral data. Other sites also bind copper but with association constants at least a factor of ten smaller. At high pH (> 8) further binding sites predominate.

scholarly journals A novel mechanism of xylan binding by a lectin-like module from Streptomyces lividans xylanase 10A

2000 ◽  
Vol 350 (3) ◽  
pp. 933-941 ◽  
Author(s):  
Alisdair B. BORASTON ◽  
Peter TOMME ◽  
Emily A. AMANDORON ◽  
Douglas G. KILBURN
Keyword(s):  
Binding Sites ◽  
Association Constant ◽  
Streptomyces Lividans ◽  
Association Constants ◽  
Carbohydrate Binding ◽  
Toxin B ◽  
Ricin Toxin ◽  
Functional Sites ◽  
Directed Mutation ◽  
Single Binding Site

The C-terminal module of xylanase 10A from Streptomyces lividans is a family 13 carbohydrate-binding module (CBM13). CBM13 binds mono- and oligo-saccharides with association constants of ≈ 1×102 M-1−1×103 M-1. It appears to be specific only for pyranose sugars. CBM13 binds insoluble and soluble xylan, holocellulose, pachyman, lichenan, arabinogalactan and laminarin. The association constant for binding to soluble xylan is (6.2±0.6)×103/mol of xylan polymer. Site-directed mutation indicates the involvement of three functional sites on CBM13 in binding to soluble xylan. The sites are similar in sequence, and are predicted to have similar structures, to the α, β and γ sites of ricin toxin B-chain, which is also in family 13. The affinity of a single binding site on CBM13 for soluble xylan is only ≈ (0.5±0.1)×103/mol of xylan. The binding of CBM13 to soluble xylan involves additive and co-operative interactions between the three binding sites. This mechanism of binding has not previously been reported for CBMs binding polysaccharides. CBM13 is the first bacterial module from family 13 to be described in detail.

scholarly journals Induction of the Cellular E2F-1 Promoter by the Adenovirus E4-6/7 Protein

2000 ◽  
Vol 74 (5) ◽  
pp. 2084-2093 ◽  
Author(s):  
Joel Schaley ◽  
Robert J. O'Connor ◽  
Laura J. Taylor ◽  
Dafna Bar-Sagi ◽  
Patrick Hearing
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transient Expression ◽  
Binding Sites ◽  
Promoter Region ◽  
Fluorescent Protein ◽  
Protein Accumulation ◽  
Promoter Regions ◽  
Single Binding Site

ABSTRACT The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with different members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. This induction of E2F DNA binding activity strongly correlates with increased E2a transcription when analyzed using virus infection and transient expression assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and stable DNA binding to the Ad5 E2a promoter region, not all of these viruses carry the inverted E2F binding site targets in their E2a promoter regions. The Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. However, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single binding site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with this notion, we show that infection with different adenovirus isolates induces the binding of E2F to an inverted configuration of binding sites present in the cellular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promoter linked to chloramphenicol acetyltransferase or green fluorescent protein reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induction of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms to induce the expression of the E2F-1 gene. The E1A proteins displace repressors of E2F activity (the Rb family members) and thus relieve E2F-1 promoter repression; the E4-6/7 protein complements this function by stably recruiting active E2F to the E2F-1 promoter to transactivate expression.

scholarly journals Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites

2001 ◽  
Vol 21 (8) ◽  
pp. 2790-2801 ◽  
Author(s):  
James F. Theis ◽  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Binding Site ◽  
Binding Sites ◽  
Origin Recognition Complex ◽  
Replication Origins ◽  
Discrete Site ◽  
Origin Function ◽  
Single Binding Site ◽  
Origin Recognition

ABSTRACT While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins inSaccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.

The determination of calcium-binding sites of human erythrocyte membranes

1984 ◽  
Vol 62 (6) ◽  
pp. 398-408 ◽  
Author(s):  
R. Blaine Moore ◽  
E. E. Dryden ◽  
D. I. C. Kells ◽  
J. F. Manery
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Calcium Binding ◽  
Plasma Membranes ◽  
Equilibrium Dialysis ◽  
Association Constants ◽  
Erythrocyte Membranes ◽  
Least Squares Regression ◽  
Calcium Binding Site ◽  
Calcium Binding Sites

Calcium binding to leaky erythrocyte plasma membranes was measured by three different procedures: Millipore filtration, equilibrium dialysis, and partition centrifugation. The curve derived from the binding equation, which best fit the means of the raw data, was used to estimate the association constants and capacities of the binding sites. A computer program (Gaushaus) which uses a nonlinear, least-squares regression protocol was also used to confirm these estimates. On the basis of these analyses we propose the presence of three classes of calcium-binding sites with the following apparent association constants and capacities: site 1, Ka = 3 × 104 M−1 and n = 30 nmol/mg protein; site 2, Ka = 3 × 103 M−1 and n = 200 nmol/mg protein; site 3, Ka = ~102 M−1 and n = ~200 nmol/mg protein. Calcium binding to erythrocyte membranes sealed in a high-salt solution showed the presence of site 3, but not site 2. The influence of phospholipids on the binding of calcium was evaluated by pretreating ghosts with phospholipase C (Clostridium welchii, EC 3.1.4.3). Treatment with this enzyme removed 80% of the total membrane phosphorus, predominantly from sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. By the method of partition centrifugation two classes of binding sites were identified by computer analysis. Their association constants and capacities are, respectively, 1.1 × 105 M−1 and 20 nmol/mg protein for site 1 and 4.4 × 103 M−1 and 200 nmol/mg protein for site 2. We speculate that calcium-binding site 1 is composed of acidic phospholipids, calcium-binding site 2 is composed of spectrin and actin, and calcium-binding site 3 is composed of sialic acid.

Differential transport properties of D-leucine and L-leucine in the archaeon, Halobacterium salinarum

2000 ◽  
Vol 46 (4) ◽  
pp. 376-382
Author(s):  
Mikiei Tanaka ◽  
Yasuo Mukohata ◽  
Seiji Yuasa
Keyword(s):  
Membrane Potential ◽  
Binding Site ◽  
Binding Sites ◽  
Halophilic Archaea ◽  
Halobacterium Salinarum ◽  
Sodium Ion ◽  
Leucine Transport ◽  
Dependent Transport ◽  
Single Binding Site ◽  
Positively Charged

The transport of D-leucine was compared with that of L-leucine in Halobacterium salinarum. When a high-outside/low-inside Na+ gradient was imposed, D-leucine as well as L-leucine accumulated in envelope vesicles, supporting the hypothesis that D-leucine is transported via a symport system along with Na+. Kinetic analyses, including inhibition experiments, indicated that both enantiomers are transported via a common carrier. However, a Hill plot indicated a single binding site for Na+ during L-leucine transport, but dual binding sites for Na+ during D-leucine transport. Furthermore, D-leucine transport was dependent on electrical membrane potential, suggesting that a transporter bound with D-leucine is positively charged. L-leucine transport was slightly, if at all, dependent on membrane potential, suggesting that a transporter bound with L-leucine is electrically neutral. These results indicate that the leucine carrier in Halobacterium salinarum translocates two moles of Na+ per mole of D-leucine, and one mole of Na+ per mole of L-leucine.Key words: D-leucine, sodium ion-dependent transport, stoichiometry, stereospecific recognition, halophilic archaea.

scholarly journals Albumin binding of photobilirubin II

1983 ◽  
Vol 213 (1) ◽  
pp. 25-29 ◽  
Author(s):  
P Meisel ◽  
K E Biebler ◽  
A Gens ◽  
K Jaehrig
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Albumin Binding ◽  
Minor Importance ◽  
High Affinity ◽  
Bilirubin Binding ◽  
Source Of Error ◽  
Single Binding Site ◽  
Site Binding

Photobilirubin II, a stereoisomer of bilirubin, binds to human serum albumin at a single binding site (K = 2.2 × 10(6)M-1), presumably the high-affinity bilirubin-binding site. Binding in the secondary (class II) binding sites is of minor importance. The results are discussed with respect to photometabolism of bilirubin and as a possible source of error in the determination of bilirubin unbound to albumin.

scholarly journals Combinatorial Regulation by a Novel Arrangement of FruA and MrpC2 Transcription Factors during Myxococcus xanthus Development

2009 ◽  
Vol 191 (8) ◽  
pp. 2753-2763 ◽  
Author(s):  
Sheenu Mittal ◽  
Lee Kroos
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Promoter Region ◽  
Developmental Process ◽  
Response Regulator ◽  
Myxococcus Xanthus ◽  
Control Cell ◽  
Cooperative Binding ◽  
Regulatory Sequence

ABSTRACT Myxococcus xanthus is a gram-negative soil bacterium that undergoes multicellular development upon nutrient limitation. Intercellular signals control cell movements and regulate gene expression during the developmental process. C-signal is a short-range signal essential for aggregation and sporulation. C-signaling regulates the fmgA gene by a novel mechanism involving cooperative binding of the response regulator FruA and the transcription factor/antitoxin MrpC2. Here, we demonstrate that regulation of the C-signal-dependent fmgBC operon is under similar combinatorial control by FruA and MrpC2, but the arrangement of binding sites is different than in the fmgA promoter region. MrpC2 was shown to bind to a crucial cis-regulatory sequence in the fmgBC promoter region. FruA was required for MrpC and/or MrpC2 to associate with the fmgBC promoter region in vivo, and expression of an fmgB-lacZ fusion was abolished in a fruA mutant. Recombinant FruA was shown to bind to an essential regulatory sequence located slightly downstream of the MrpC2-binding site in the fmgBC promoter region. Full-length FruA, but not its C-terminal DNA-binding domain, enhanced the formation of complexes with fmgBC promoter region DNA, when combined with MrpC2. This effect was nearly abolished with fmgBC DNA fragments having a mutation in either the MrpC2- or FruA-binding site, indicating that binding of both proteins to DNA is important for enhancement of complex formation. These results are similar to those observed for fmgA, where FruA and MrpC2 bind cooperatively upstream of the promoter, except that in the fmgA promoter region the FruA-binding site is located slightly upstream of the MrpC2-binding site. Cooperative binding of FruA and MrpC2 appears to be a conserved mechanism of gene regulation that allows a flexible arrangement of binding sites and coordinates multiple signaling pathways.

scholarly journals The low C5 convertase activity of the C4A6 allotype of human complement component C4

1989 ◽  
Vol 261 (3) ◽  
pp. 743-748 ◽  
Author(s):  
T Kinoshita ◽  
A W Dodds ◽  
S K A Law ◽  
K Inoue
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Alternative Pathway ◽  
Covalent Attachment ◽  
Haemolytic Activity ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Covalently Linked

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.

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