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.

Tuning of Electronic Properties in Conducting Polymers

2001 ◽  
Vol 66 (8) ◽  
pp. 1208-1218 ◽  
Author(s):  
Guofeng Li ◽  
Mira Josowicz ◽  
Jiří Janata
Keyword(s):  
Hydrogen Bonding ◽  
Binding Site ◽  
Binding Sites ◽  
Polymer Chains ◽  
Electronic Transitions ◽  
Weakly Bound ◽  
Ordered Structures ◽  
Doped Polymer ◽  
Positively Charged ◽  
Repeated Cycling

Structural and electronic transitions in poly(thiophenyleneiminophenylene), usually referred to as poly(phenylenesulfidephenyleneamine) (PPSA) upon electrochemical doping with LiClO4 have been investigated. The unusual electrochemical behavior of PPSA indicates that the dopant anions are bound in two energetically different sites. In the so-called "binding site", the ClO4- anion is Coulombically attracted to the positively charged S or N sites on one chain and simultaneously hydrogen-bonded with the N-H group on a neighboring polymer chain. This strong interaction causes a re-organization of the polymer chains, resulting in the formation of a networked structure linked together by these ClO4- Coulombic/hydrogen bonding "bridges". However, in the "non-binding site", the ClO4- anion is very weakly bound, involves only the electrostatic interaction and can be reversibly exchanged when the doped polymer is reduced. In the repeated cycling, the continuous and alternating influx and expulsion of ClO4- ions serves as a self-organizing process for such networked structures, giving rise to a diminishing number of available "non-binding" sites. The occurrence of these ordered structures has a major impact on the electrochemical activity and the morphology of the doped polymer. Also due to stabilization of the dopant ions, the doped polymer can be kept in a stable and desirable oxidation state, thus both work function and conductivity of the polymer can be electrochemically controlled.

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.

scholarly journals One Intact Transmembrane Substrate Binding Site Is Sufficient for the Function of the Homodimeric Type I ATP-Binding Cassette Importer for Positively Charged Amino Acids Art(MP) 2 of Geobacillus stearothermophilus

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Johanna Heuveling ◽  
Heidi Landmesser ◽  
Erwin Schneider
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Atp Binding ◽  
Content Type ◽  
Charged Amino Acids ◽  
Substrate Binding Sites ◽  
Positively Charged ◽  
Atp Binding Cassette

ABSTRACT ATP-binding cassette (ABC) transport systems comprise two transmembrane domains/subunits that form a translocation path and two nucleotide-binding domains/subunits that bind and hydrolyze ATP. Prokaryotic canonical ABC import systems require an extracellular substrate-binding protein for function. Knowledge of substrate-binding sites within the transmembrane subunits is scarce. Recent crystal structures of the ABC importer Art(QN) 2 for positively charged amino acids of Thermoanerobacter tengcongensis revealed the presence of one substrate molecule in a defined binding pocket in each of the transmembrane subunits, ArtQ (J. Yu, J. Ge, J. Heuveling, E. Schneider, and M. Yang, Proc Natl Acad Sci U S A 112:5243–5248, 2015, https://doi.org/10.1073/pnas.1415037112 ). This finding raised the question of whether both sites must be loaded with substrate prior to initiation of the transport cycle. To address this matter, we first explored the role of key residues that form the binding pocket in the closely related Art(MP) 2 transporter of Geobacillus stearothermophilus , by monitoring consequences of mutations in ArtM on ATPase and transport activity at the level of purified proteins embedded in liposomes. Our results emphasize that two negatively charged residues (E153 and D160) are crucial for wild-type function. Furthermore, the variant Art[M(L67D)P] 2 exhibited strongly impaired activities, which is why it was considered for construction of a hybrid complex containing one intact and one impaired substrate-binding site. Activity assays clearly revealed that one intact binding site was sufficient for function. To our knowledge, our study provides the first biochemical evidence on transmembrane substrate-binding sites of an ABC importer. IMPORTANCE Canonical prokaryotic ATP-binding cassette importers mediate the uptake of a large variety of chemicals, including nutrients, osmoprotectants, growth factors, and trace elements. Some also play a role in bacterial pathogenesis, which is why full understanding of their mode of action is of the utmost importance. One of the unsolved problems refers to the chemical nature and number of substrate binding sites formed by the transmembrane subunits. Here, we report that a hybrid amino acid transporter of G. stearothermophilus , encompassing one intact and one impaired transmembrane binding site, is fully competent in transport, suggesting that the binding of one substrate molecule is sufficient to trigger the translocation process.

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 Membrane-Potential-Sensitive, Na+-Independent Lysine Transport by Lobster Hepatopancreatic Brush Border Membrane Vesicles

1987 ◽  
Vol 127 (1) ◽  
pp. 373-387
Author(s):  
GREGORY A. AHEARN ◽  
LAUREL P. CLAY
Keyword(s):  
Membrane Potential ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Acidic Ph ◽  
S Uptake ◽  
Apparent Diffusion ◽  
Leucine Transport ◽  
Dependent Transport

Transport of l-[3H]lysine by epithelial brush border membrane vesicles (BBMV) of lobster hepatopancreas, formed by a magnesium precipitation technique, was insensitive to transmembrane gradients of Na+, K+, TMA+ or H+. Apparent initial lysine entry rates (15 s uptake) and extent of amino acid accumulation against a concentration gradient (overshoot) were both stimulated by transmembrane anion gradients according to the following sequence: SCN− &gt; Cl− &gt; gluconate−. The magnitude of this anion-gradient-dependent transport was significantly increased by bilateral acidic pH. Lysine transport at acidic pH strongly responded to transmembrane potential developed by addition of valinomycin to K+-loaded vesicles, or was markedly reduced if K+-equilibrated vesicles were incubated with the ionophore in the presence of an inwardly directed SCN− gradient. Lysine influx occurred by the combination of at least one carrier process and ‘apparent diffusion’. l-Arginine, l-alanine and l-leucine, added to the external medium, were all strong inhibitors of lysine influx. The first two were competitive inhibitors of lysine entry, while the latter was non-competitive in effect. These results suggest that lysine, arginine and alanine may share a common, Na+-independent, membrane-potential-sensitive transport mechanism in lobster BBMV. Leucine transport may occur in these membranes by a separate agency.

scholarly journals Structure and Function of Flavivirus NS5 Methyltransferase

2007 ◽  
Vol 81 (8) ◽  
pp. 3891-3903 ◽  
Author(s):  
Yangsheng Zhou ◽  
Debashish Ray ◽  
Yiwei Zhao ◽  
Hongping Dong ◽  
Suping Ren ◽  
...  
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Binding Pocket ◽  
Wild Type ◽  
Terminal Portion ◽  
Novel Target ◽  
And Function ◽  
Methylation Activity ◽  
Single Binding Site ◽  
Positively Charged

ABSTRACT The plus-strand RNA genome of flavivirus contains a 5′ terminal cap 1 structure (m7GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2′-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA → m7GpppA → m7GpppAm. The 2′-O methylation can be uncoupled from the N-7 methylation, since m7GpppA-RNA can be readily methylated to m7GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 Å resolution showed a single binding site for S-adenosyl-l-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2′-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2′-O cap methylations require distinct buffer conditions and different side chains within the K61-D146-K182-E218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2′-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.

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.

scholarly journals The effective opening of nicotinic acetylcholine receptors with single agonist binding sites

2011 ◽  
Vol 137 (4) ◽  
pp. 369-384 ◽  
Author(s):  
Dustin K. Williams ◽  
Clare Stokes ◽  
Nicole A. Horenstein ◽  
Roger L. Papke
Keyword(s):  
Binding Site ◽  
Nicotinic Acetylcholine Receptors ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Xenopus Laevis Oocytes ◽  
Wild Type ◽  
Muscle Type ◽  
Net Charge ◽  
Single Binding Site

We have identified a means by which agonist-evoked responses of nicotinic receptors can be conditionally eliminated. Modification of α7L119C mutants by the sulfhydryl reagent 2-aminoethyl methanethiosulfonate (MTSEA) reduces responses to acetylcholine (ACh) by more than 97%, whereas corresponding mutations in muscle-type receptors produce effects that depend on the specific subunits mutated and ACh concentration. We coexpressed α7L119C subunits with pseudo wild-type α7C116S subunits, as well as ACh-insensitive α7Y188F subunits with wild-type α7 subunits in Xenopus laevis oocytes using varying ratios of cRNA. When mutant α7 cRNA was coinjected at a 5:1 ratio with wild-type cRNA, net charge responses to 300 µM ACh were retained by α7L119C-containing mutants after MTSEA modification and by the ACh-insensitive Y188F-containing mutants, even though the expected number of ACh-sensitive wild-type binding sites would on average be fewer than two per receptor. Responses of muscle-type receptors with one MTSEA-sensitive subunit were reduced at low ACh concentrations, but much less of an effect was observed when ACh concentrations were high (1 mM), indicating that saturation of a single binding site with agonist can evoke strong activation of nicotinic ACh receptors. Single-channel patch clamp analysis revealed that the burst durations of fetal wild-type and α1β1γδL121C receptors were equivalent until the α1β1γδL121C mutants were exposed to MTSEA, after which the majority (81%) of bursts were brief (≤2 ms). The longest duration events of the receptors modified at only one binding site were similar to the long bursts of native receptors traditionally associated with the activation of receptors with two sites containing bound agonists.

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